In addition to carrying genetic information, nucleic acids can adopt complex three-dimensional structures. These three-dimensional structures are capable of specific recognition of target molecules and, furthermore, of catalyzing chemical reactions. Nucleic acids will thus provide candidate detection molecules for diverse target molecules, including those which that do not naturally recognize or bind to DNA or RNA.
A nucleic acid which binds to a non-nucleic acid target molecule through non-Watson-Crick base pairing is termed an aptamer. In aptamer selection, combinatorial libraries of oligonucleotides are screened in vitro to identify oligonucleotides, or aptamers, which bind with high affinity to pre-selected targets. Both small biomolecules (e.g., amino acids, nucleotides, NAD, S-adenosyl methionine, chloramphenicol), and large biomolecules (thrombin, Ku, DNA polymerases) are effective targets for aptamers. Aptamer biosensors have been used to detect specific analyte molecules. For example, fluorescently labeled anti-thrombin aptamers attached to a glass surface have been used to directly detect the presence of thrombin proteins in a sample by detecting changes in the optical properties of the aptamers (Potyrailo, et al., 1998). In this method continuous binding of thrombin to the labeled aptamer is requisite for detection to occur, since the concentration of thrombin in a test sample is monitored by directly detecting fluorescent emission of the aptamer-ligand complex. Another method of detecting binding of a ligand to an aptamer has also been described which relies on the use of fluorescence-quenching pairs whose fluorescence is sensitive to changes in secondary structure of the aptamer upon ligand binding (Stanton, et al. 2000) to form a fluorescent aptamer-ligand complex, and again, continuous binding of the ligand to the aptamer is required for signal generation and, hence, for detection to occur. A limitation with this type of aptamer-derived biosensor is that ligand-mediated changes in secondary structure were engineered into the aptamer molecule via a laborious engineering process in which four to six nucleotides were added to the 5′ end of the aptamer that was complementary to the bases at the 3′ end of the thrombin binding region. In the absence of thrombin, this structure forms a stem loop structure, while it forms a G-quartet structure in the presence of thrombin. Fluorescent and quenching groups attached to the 5′ and 3′ end signal this change. In aptamer-based detection without the use of amplification steps, assay sensitivity and hence the limit of detection is set empirically by the affinity of the aptamer-ligand complex, the KD value. Using aptamer-based sensor molecules one can detect analyte binding in both solution (homogeneous) and on solid supports (heterogeneous).
Other ligand detection methods known in the art are based upon antibody binding. Similar to the aptamer-based methods, antibody-based detection requires continuous ligand binding and ligand-antibody complex formation for the generation of a detectable signal. In addition, antibody methods such as ELISA or competitive RIA, while robust, are restricted in utility because these methods require that heterogeneous assay conditions be employed: 1] detection is done on a solid surface; 2] in most applications both a capture antibody and detection antibody are required; 3] for ELISA-based protein detection methods, the antibodies must recognize the folded, native structure of the protein that is present in cell or tissue isolates and; 4] antibody and protein based detection methods have not been described for intracellular or in vivo based analyte detection. That antibodies have not been employed for intracellular and in vivo based detection of proteins, drugs or metabolites is due to several technological factors. First monoclonal antibody fragments are unstable and do not fold properly when expressed as intracellular protein molecules. Second, intracellular detection requires homogeneous assay formats and these solution-based detection methods require the sensor to have a ligand sensing or modulation domain coupled directly to a catalytic or signal generating region of an enzyme or catalytic biomolecule. A fundamental and important consequence of the limitations of antibody-based detection methods is that they can not function as a universal reagent for all assays and tests that can be employed in drug discovery and development. These assays include 1] the initial discovery of a drug target through protein or metabolite profiling, 2] the subsequent use of that same drug target in the discovery of drug leads through high throughput screening and, 3] the optimization of drug leads against that same drug target through an evaluation of lead efficacy in mechanistic cellular and in vivo animal assays.
To streamline the drug discovery and development process and improve the efficiency of evaluating drug targets and drug leads, detection reagents are needed that can function in a context-independent manner. Needed then is a molecular sensor that can function in multiple assay environments and in multiple assay formats. The present invention is generally drawn to nucleic acid molecular sensors that can function in environments and formats that includes but are not limited to solution-based detection (homogeneous in vitro biochemical assays or in vivo cellular and animal assays), chip-based (heterogeneous in vitro assays on solid surfaces), and assays in complex biological isolates from blood plasma, cell lysates or tissue extracts.
Nucleic acid-based detection schemes have exploited the ligand-sensitive catalytic properties of some nucleic acids, e.g., such as ribozymes. Ribozyme-based prototype nucleic acid sensor molecules have been designed both by engineering and by in vitro selection methods. Engineering methods exploit the apparently modular nature of RNA structures; these sensors couple molecular recognition to signaling by simply joining individual target-modulation and catalytic RNA domains through a double-stranded or partially double-stranded RNA linker. ATP sensors, for example, were created by appending the previously-selected, ATP-binding aptamer-derived sequences (Sassanfar and Szostak, 1993) to either the self-cleaving hammerhead ribozyme (Tang and Breaker, 1997) or the L1 self-ligating ribozyme (Robertson and Ellington 2000). Robertson and Ellington (2000) have demonstrated that the enzymatic activity of a ligase ribozyme (derived from the L1 ligase described in Robertson and Ellington (1999)) can be modulated by a small molecule ligand, or small molecule target recognition. In this case, the ligase ribozyme can be employed as a nucleic acid sensor molecule and used to detect the presence and level of its cognate ligand by monitoring the ligation of a small, labeled second oligonucleotide substrate on to the ribozyme. A distinct feature of this detection method is that the actual detection event, e.g., monitoring oligonucleotide substrate ligation to the ribozyme, occurs after the ligand interacts with the nucleic sensor molecule. Hence, unlike antibody, or aptamer based detection methods, the ribozyme-based ligand detection method of Robertson and Ellington does not require continuous binding of the ligand to the sensor molecule in order to generate a detectable signal. In a complementary approach, radiolabeled hammerhead ribozymes which undergo cleavage upon binding to a ligand, have been used to detect ligand by monitoring the release of the label from the ribozyme (Soukup, et al., 2000, and Breaker, 1998). Limitations of the use of ligand modulated hammerhead ribozymes described by Soukup, et al., 2000, and Breaker, 1998 include: 1] the need for a two-step detection method for determining the enzymatic activity of the surface-immobilized hammerhead-derived sensors; 2] the need for radiometric determination of hammerhead activity in both solution and solid-surface based assay formats; 3] the need for significant chemical and structural modification of the hammerhead-based biosensor to render them suitable for optical based detection methods.
Another limitation of the engineering method to sensor generation is that it has been generally thought not to be applicable to the development of protein-dependent ribozymes. Robertson and Ellington (2001) describe their own efforts to extend this methodology to the identification of protein and peptide-dependent ribozymes, but state that simply appending aptamer-derived sequences to the catalytic domain of the L1 ligase at stem C, yields little or no target dependent modulation. Furthermore, the authors state that the “principles required for engineering protein-dependent ribozymes must be fundamentally different from those for identifying ribozymes dependent on small-molecules.” Hence, in order to identify protein and peptide dependent ribozymes, Ellington and Robertson undertook a laborious in vitro selection process which involved randomization of the catalytic core of the L1 ligase coupled with multiple rounds of positive and negative selection. Ellington and Roberts (2000) describe several limitations of the ligase-derived sensors that they developed. First, the nucleoprotein enzymes developed by Ellington and Robertson (2000) required a laborious in vitro selection process to identify peptide and protein dependent ligases. Secondly, Ellington and Roberts (1999) describe a region of the L1 ligase that is required for allosteric ribozyme function, termed the effector oligonucleotide binding domain. It was postulated that the effector oligonucleotide binding domain of the ligase formed complementary base pairing interactions with the oligonucleotide substrate binding site, driving the ribozyme into an inactive conformation. The effector oligonucleotide, when added to the L1 ligase activates (kact) the enzyme by over 10,000 fold over the L1 ligase reaction measure in the absence of effector (kunact). Hence, the native L1-ligase has a switch factor (kact/kunact) greater than 10,000, which determines the sensitivity of a ribozyme-based detection method. When the effector oligonucleotide binding domain of the L1 ligase is deleted, the ligase activity of the deletion mutant is only 3–5 fold lower than the ligase activity of L1 ligase with the effector oligonucleotide bound to the effector oligonucleotide binding domain (Ellington and Robertson (1999). This indicates that L1 ligases deleted of the effector oligonucleotide binding domain may not be not subject to further allosteric regulation. Hence, a hindrance to the development of L1 ligase-based biosensor technology is the lack of a general method for the generation of biosensors that can work in multiple assay and detection formats required of solution-based and chip-based biosensors and, those that can work in multiplexed formats and in complex biological extracts.